RNA participates in a wide range of biological processes. In addition to its role in information transfer in the genetic code, it can act as a structural scaffold, a catalytic enzyme, or a regulatory signal. In the clinical context it is both a target for drug design and a tool for regulating gene function. Much of RNA function is driven by its interaction with metal ions and metals play a pivotal role in maintaining RNA's structure and promoting the catalytic mechanism. The principle goals of this proposal are to develop new modalities to characterize metal RNA interactions and the chemical reactions they promote. The methods involve the use of Raman spectroscopy to probe RNA in solution and Raman microscopy to study reactions in single crystals of RNA. The solution studies will use small RNA fragments, less than 40 nucleotides, in order to characterize a metal cation binding at a single phosphate group along with conformational changes in the adjacent phosphodiester backbone. The single crystal studies center on four RNAs, the Hepatitis Delta Virus (HDV) ribozyme, the P4-P6 domain and Twort ribozyme from group I introns, and phenylalanine tRNA. Magnesium binding sites in the crystal structures that have been characterized by X-ray crystallography are being used to provide the Raman signatures of -PO2- groups making inner sphere contacts with magnesium. The low frequency Raman spectral region provides the spectra, and thus chemical information, associated with the bound metals themselves. A major theme is to use Raman crystallography, combined with X-ray crystallography to provide unambiguous answers to outstanding issues of mechanism. For example, for HDV crystals we label active site G1and U-1 groups with 13C and 15N isotopes to allow us to follow these residues as catalytic parameters such as pH and Mg concentration are changed. These observations will be extended by using the labeling approach to define the chemistry of the key active site groups as the chemical reaction is being carried out in the crystal. An overarching aspect of this proposal is its interdisciplinary nature that involves collaborators at three locations. The Raman spectroscopy and Raman crystallography are undertaken in the Carey laboratory at CWRU, while the expertise and material for solution studies is provided by Drs. Mike Harris and Eric Christian from CWRU and Dr. Kwaku Dayie at the Cleveland Clinic. The work on RNA crystals is based on collaborations with Dr. Phil Bevilacqua at Penn State and Dr. Barbara Golden at Purdue. PUBLIC HEALTH RELEVANCE: Although RNA is a close relative of DNA it is far more versatile and plays many roles in the life of the cell. Thus, an understanding of the properties of RNA lies at the heart of our knowledge of the processes of life and to be able to control them when malfunction leads to disease states. The present proposal sets out to determine how RNA acts as a catalyst to bring about chemical reactions. The research involves following chemical reactions in very small RNA crystals using a laser light scattering technique.